The centralization of food processing in large plants supplying millions of pounds of food products has aggravated the problems associated with microbial contamination of the products. When a production line becomes contaminated, the health of large numbers of people is threatened. In addition, the centralization of processing increases the time period between the processing of the food product and the final consumption of that product. Since bacteria continue to grow during this time period, a marginally contaminated product can become unfit for consumption by the time it reaches the consumer.
Accordingly, there is an increasing need for systems that detect contamination at the plant in time to prevent shipment of a contaminated product. Ideally, such a system would detect contamination in time to shut down a production line before large quantities of product are contaminated. Since bacteria can grow rapidly on food products such as ground meat, such an assay system must be able to detect contamination at very low levels, typically 100 to 1000 bacteria per gram of ground meat.
Assay systems that can quickly detect microorganisms based on immunological techniques have been known for some time. Immunoassay techniques are based on the ability of antibodies to form complexes with the corresponding antigens. This property of highly specific molecular recognition of antigens by antibodies leads to high selectivity of assays based on immune principles. The high affinity of antigen-antibody interactions allows very small quantities of microorganisms to be determined. In addition, microorganisms are typically classified according to the antigens on the organism's surface; hence, immunological assays also yield results that provide the identity of the contaminating organism as well as the level of contamination.
Immunoassay techniques are used mainly in clinical analyses and medical diagnostics. Immunoassay techniques could, in principle, be utilized in many non-clinical applications if assay systems that were better adapted for field operating conditions were available. Conventional immunoassay techniques (such as ELISA, immunoblot, immunoagglutination) can be used only in specially equipped laboratories and require technically trained personnel. These assays are difficult to conduct in the non-laboratory conditions typically encountered in field settings or food processing lines.
During the last few years, a significant number of publications have dealt with alternative immunoassay techniques. The development of alternative immunoassay techniques aims in most cases at improvements in performance of conventional immuno-analysis to decrease the analysis time, increase assay sensitivity, and simplify and automate assay procedures. The basic principles of the alternative immunoassay methods are the same as for conventional immunoassay techniques in that these alternative assays are also based on the detection of antigen-antibody interaction. Frequently the term `biosensor` or `immunosensor` is used to label an immunoassay system that is an alternative to a conventional assay system, developed with automated data acquisition.
In general, an immuno-sensor (biosensor) consists of a signal transducer and a biochemically interactive system employing principles of biological molecular recognition. Based on the nature of the physical detection used in the transducer, immuno-sensing systems can be classified as optical, gravimetric and electrochemical. In optical transducers, detection is based on light-sensitive elements. The optical signal detection can be conducted by spectrophotometric, spectrofluorimetric, hemiluminometric, reflectometric or other related techniques.
Gravimetric transducers are based on sensitive detection of mass changes following antigen-antibody complex formation. Piezoelectric detectors are typically based on acoustical resonators having resonant frequencies that are altered by the change in mass of a layer which is in contact with the resonator. This layer typically includes one member of an antigen-antibody complex. When the other member attaches to the layer, the resonance frequency shifts. These transducers cannot distinguish between specific binding and non-specific binding.
Electrochemical transducers are based on detection of changes in electro transfer caused by the immuno-interaction. In particular, this detection is brought about using amperometric, poteniometric, conductometric (at constant voltage) or impedimetric (at alternative voltage) devices.
Flow-injection principles can be used to enhance the efficiency of the immuno-interaction. Prior art flow-injection immuno-sensing systems arc based on a principle of displacement. In this case, the immunoassay system is arranged as a column containing immobilized antibodies. The column is saturated with a solution containing a labeled antigen. After antigen-antibody interaction has occurred, the column contains a solid carrier with immobilized antibody-labeled antigen complexes. The affinity of antibodies for labeled antigens is usually significantly lower than their affinity for unlabeled (free) antigen due to stearic factors. Therefore, injection of free antigen into the column results in displacement of the labeled antigen by the unlabeled antigen. Labeled antigen is then detected at the outlet of the column. A similar scheme can be realized based on the use of immobilized antigen. In this case, injection of the analyte leads to replacement of the antibody-conjugated complex. Flow-injection immunoassay systems based on displacement schemes for real time (two-three minutes) determination of a number haptens have been reported by Liegler, et al. in U.S. Pat. No. 5,183,740. However, these flow-injection schemes have substantially less sensitivity than the conventional assay systems.
Traditional immuno-analysis schemes (competitive binding and `sandwich` schemes) are also employed in flow-injection immunoassay systems. In these cases, the problem of column regeneration is an important issue. The problem associated with the necessity to renew the immuno-sorbent in flow-injection systems can be solved by development of disposable immuno-columns. While such sensors solve the problems associated with disposability in a clinical laboratory setting, these sensors are less than optimal in the food production line setting in which multiple infective agent assays must be performed on samples that include biological material that clogs the sensors and exhibit non-specific binding for the sensor material.
Broadly, it is the object of the present invention to provide an improved immunoassay apparatus.
It is a further object of the present invention to provide a disposable flow-immunoassay apparatus that is adapted for use in the food processing environment.
It is yet another object of the present invention to provide an assay system that can simultaneously detect multiple infective agents.
These and other objects of the present invention will become apparent to those skilled in the art from the following detailed description of the invention and the accompanying drawings.